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1. A Tunisian family with a novel mutation in the gene CYP4F22 for lamellar ichthyosis and co‐occurrence of hearing loss in a child due to mutation in the SLC26A4 gene

   https://doi.org/10.1111/ijd.14452  

   Sayeb, Marwa ; Riahi, Zied ; Laroussi, Nadia ; Bonnet, Crystel ; Romdhane, Lilia ; Mkaouar, Rahma ; Zaouak, Anissa ; Marrakchi, Jihene ; Abdessalem, Ghaith ; Messaoud, Olfa ; et al ( April 2019 , International Journal of Dermatology)

   Co‐occurrence of two genetic diseases is challenging for accurate diagnosis and genetic counseling. The recent availability of whole exome sequencing (WES) has dramatically improved the molecular diagnosis of rare genetic diseases in particular in consanguineous populations.

   We report here on a consanguineous family from Southern Tunisia including three members affected with congenital ichthyosis. The index case had a hearing loss (HL) and ichthyosis and was primarily suspected as suffering from keratitis‐ichthyosis‐deafness (KID) syndrome.WESwas performed for the index case, and all members of the nuclear family were sequenced (Sanger method).

   TheWESapproach allowed the identification of two strong candidate variants in two different genes; a missense mutation c.1334T>G (p.Leu445Trp) in exon 11 ofSLC26A4gene, associated with isolatedHLand a novel missense mutation c.728G>T (p.Arg243Leu) in exon 8 ofCYP4F22gene likely responsible for ichthyosis. These two mutations were predicted to be pathogenic by three pathogenicity prediction softwares (Scale‐Invariant Feature Transform [SIFT], Polymorphism Phenotyping [PolyPhen], Mutation Taster) to underlie theHLand ichthyosis, respectively.

   The present study raises awareness about the importance of familial history for accurate diagnosis of syndromic genetic diseases and differential diagnosis with co‐occurrence of two distinct clinical entities. In addition, in countries with limited resources,WESsequencing for a single individual provides a cost effective tool for molecular diagnosis confirmation and genetic counseling.

    

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2. CRISPR /Cas9 mutagenesis in Volvox carteri

   https://doi.org/10.1111/tpj.14149  

   Ortega‐Escalante, José A. ; Jasper, Robyn ; Miller, Stephen M. ( January 2019 , The Plant Journal)

   Volvox carteriand other volvocine green algae comprise an excellent model for investigating developmental complexity and its origins. Here we describe a method for targeted mutagenesis inV. carteriusingCRISPR/Cas9 components expressed from transgenes. We usedV. carterinitrate reductase gene (nitA) regulatory sequences to conditionally expressStreptococcus pyogenesCas9, andV. carteriU6RNAgene regulatory sequences to constitutively express single‐guideRNA(sgRNA) transcripts.Volvox carteriwas bombarded with both Cas9 vector and one of several sgRNAvectors programmed to target different test genes (glsA,regAandinvA), and transformants were selected for expression of a hygromycin‐resistance marker present on the sgRNAvector. Hygromycin‐resistant transformants grown with nitrate as sole nitrogen source (inducing fornitA) were tested for Cas9 and sgRNAexpression, and for the ability to generate progeny with expected mutant phenotypes. Some transformants of a somatic regenerator (Reg) mutant strain receiving sgRNAplasmid withglsAprotospacer sequence yielded progeny (at a rate of ~0.01%) with a gonidialess (Gls) phenotype similar to that observed for previously describedglsAmutants, and sequencing of theglsAgene in independent mutants revealed short deletions within the targeted region ofglsA, indicative of Cas9‐directed non‐homologous end joining. Similarly, bombardment of a morphologically wild‐type strain with the Cas9 plasmid and sgRNAplasmids targetingregAorinvAyieldedregAandinvAmutant transformants/progeny, respectively (at rates of 0.1–100%). The capacity to make precisely directed frameshift mutations should greatly accelerate the molecular genetic analysis of development inV. carteri, and of developmental novelty in the volvocine algae.

    

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3. Nuclear‐encoded sigma factor 6 ( SIG 6) is involved in the block of greening response in Arabidopsis thaliana

   https://doi.org/10.1002/ajb2.1423  

   Alameldin, Hussien F. ; Oh, Sookyung ; Hernandez, Alexandra P. ; Montgomery, Beronda L. ( January 2020 , American Journal of Botany)

   Light is critical in the ability of plants to accumulate chlorophyll. When exposed to far‐red (FR) light and then grown in white light in the absence of sucrose, wild‐type seedlings fail to green in a response known as theFRblock of greening (BOG). This response is controlled by phytochrome A through repression of protochlorophyllide reductase‐encoding (POR) genes byFRlight coupled with irreversible plastid damage. Sigma (SIG) factors are nuclear‐encoded proteins that contribute to plant greening and plastid development through regulating gene transcription in chloroplasts and impacting retrograde signaling from the plastid to nucleus.SIGs are regulated by phytochromes, and the expression of someSIGfactors is reduced in phytochrome mutant lines, including phyA. Given the association of phyA with theFR BOGand its regulation ofSIGfactors, we investigated the potential regulatory role ofSIGfactors in theFR BOGresponse.

   We examinedFR BOGresponses insigmutants, phytochrome‐deficient lines, and mutant lines for several phy‐associated factors. We quantified chlorophyll levels and examined expression of keyBOG‐associated genes.

   Among sixsigmutants, only thesig6 mutant significantly accumulated chlorophyll afterFR BOGtreatment, similar to thephyAmutant.SIG6 appears to control protochlorophyllide accumulation by contributing to the regulation of tetrapyrrole biosynthesis associated with glutamyl‐tRNAreductase (HEMA1) function, select phytochrome‐interacting factor genes (PIF4andPIF6), andPENTA1, which regulatesPORAmRNAtranslation afterFRexposure.

   Regulation ofSIG6plays a significant role in plant responses toFRexposure during theBOGresponse.

    

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4. The Sarracenia alata pitcher plant system and obligate arthropod inquilines should be considered an evolutionary community

   https://doi.org/10.1111/jbi.13498  

   Satler, Jordan D. ; Carstens, Bryan C. ( January 2019 , Journal of Biogeography)

   TheSarracenia alatapitcher plant and inquiline species comprise an ecological community. These inquilines span the continuum in their ecological association with the host pitcher plant, from species that contain little‐to‐no interaction with the plant to species that are completely dependent on the plant for their entire life cycle. We are interested in testing if degree of ecological dependence is positively correlated with a shared evolutionary history, and in identifying members of this community that display concordant phylogeographic structure.

   Southeastern United States.

   We collected genome‐wide sequence data from a set of arthropods that are ecologically associated with the plant to estimate comparative phylogeographic patterns among the species. We estimated species tree distributions from biallelic unlinkedSNPdata and used phylogeographic concordance factors (PCFs) to test degree of phylogeographic concordance among community members. In addition, for each species we conducted an analysis of molecular variance and calculatedF_STvalues to identify population genetic structure across the landscape, and compared these traditional values to the tree‐based approach.

   Obligate members of the pitcher plant community display concordant phylogeographic patterns, suggesting their ecological dependence has manifested itself into a shared evolutionary history. In contrast, two spider species do not contain significant population genetic structure or similar phylogeographic histories, highlighting their loose association with the host pitcher plant.

   TheS. alatapitcher plant system should be considered an evolutionary community, where multiple members sharing strong ecological interactions also display concordant phylogeographic structure. This work demonstrates thatPCFs provide an important quantitative measure into assessing community structure and illustrates how simulations can be used to assess significance of shared patterns of phylogeographic structure across the landscape.

    

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5. Accurate measurement of transgene copy number in crop plants using droplet digital PCR

   https://doi.org/10.1111/tpj.13517  

   Collier, Ray ; Dasgupta, Kasturi ; Xing, Yan‐Ping ; Hernandez, Bryan Tarape ; Shao, Min ; Rohozinski, Dominica ; Kovak, Emma ; Lin, Jeanie ; de Oliveira, Maria Luiza P. ; Stover, Ed ; et al ( April 2017 , The Plant Journal)

   Genetic transformation is a powerful means for the improvement of crop plants, but requires labor‐ and resource‐intensive methods. An efficient method for identifying single‐copy transgene insertion events from a population of independent transgenic lines is desirable. Currently, transgene copy number is estimated by either Southern blot hybridization analyses or quantitative polymerase chain reaction (qPCR) experiments. Southern hybridization is a convincing and reliable method, but it also is expensive, time‐consuming and often requires a large amount of genomicDNAand radioactively labeled probes. Alternatively,qPCRrequires lessDNAand is potentially simpler to perform, but its results can lack the accuracy and precision needed to confidently distinguish between one‐ and two‐copy events in transgenic plants with large genomes. To address this need, we developed a droplet digitalPCR‐based method for transgene copy number measurement in an array of crops: rice, citrus, potato, maize, tomato and wheat. The method utilizes specific primers to amplify target transgenes, and endogenous reference genes in a single duplexed reaction containing thousands of droplets. Endpoint amplicon production in the droplets is detected and quantified using sequence‐specific fluorescently labeled probes. The results demonstrate that this approach can generate confident copy number measurements in independent transgenic lines in these crop species. This method and the compendium of probes and primers will be a useful resource for the plant research community, enabling the simple and accurate determination of transgene copy number in these six important crop species.

    

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